Thymol

Hyalomma lusitanicum engorged females were collected from red deer in Ciudad Real (central Spain) and maintained in an incubator (22–24 °C and 80% relative humidity (RH) until oviposition and egg hatching.
Tests were conducted as previously described [63 (link)]. Briefly, 50 µL of test solution was added to 25 mg of powdered cellulose at different concentrations (initial concentration of 40 or 20 µg/mg for extracts and pure compounds respectively) and the solvent evaporated. Each test consisted of 20 active larvae, at least 6 weeks old, and was replicated three times. Negative (cellulose) and positive (thymol Sigma Aldrich at 20 µg/mg) controls were also used. The ticks and cellulose were placed in glass tubes and carefully mixed by rotating the tube several times to ensure full tick-cellulose contact and kept in a stove at the conditions described [63 (link)].
Dead (not moving) ticks were counted after 24 h with a binocular magnifying glass. Larvicidal activity data are presented as percent mortality corrected according to the Schneider-Orelli formula [62 ]. thymol (Sigma-Aldrich) was included as a positive control with LD₅₀ and LD₉₀ values of 2.94 and 6.16 µg/mg.

Thymol was purchased from Sigma-Aldrich, India. Ten milligrams of Thymol dissolved in 1 ml of methanol was used as stock solution (10 mg/ml). Methanol alone was used as vehicle control in all the assays.

For evaluating thymol’s genotoxic effects, five experimental sets of cells were established. In the negative control group, cells were treated only with the PBS solution, whereas the cells in the positive control group were treated with 100 µM H₂O₂. In three thymol-treated groups, the following experimental concentrations of thymol were tested: 10, 100 and 1000 µg/mL. For the purpose of the experiment, we used thymol with a purity of ≥98.5% (Sigma-Aldrich, St. Louis, MO, USA, Product Number T0501, CAS 89-83-8).
To determine possible antigenotoxic effects of thymol, in addition to negative (incubated in PBS only) and positive control (treated with 100 µM H₂O₂), three more groups were established in which AmE-711 cells were co-treated with 100 µM H₂O₂ and thymol in concentrations of 10 µg/mL (group P10), 100 µg/mL (group P100) and 1000 µg/mL (group P1000). All experimental groups were incubated for 30 min at 32 °C.